Methods of treating SARS-CoV-2 infections

This application is a National Stage Application filed under 35 U.S.C. 371 of International Application No. PCT/US2021/026302, filed Apr. 8, 2021, which claims priority to and the benefit of U.S. Provisional Patent Application No. 63/008,486, filed Apr. 10, 2020 and U.S. Provisional Patent Application No. 63/062,599, filed Aug. 7, 2020, the disclosure of which is incorporated herein by reference in its entirety.

The disclosure is directed to methods of using dantrolene or a dantrolene prodrug, or a pharmaceutically acceptable salt thereof, to treat COVID-19 and SARS-CoV-2 infections.

Worldwide progression of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to a rapid increase in the number of cases of Coronavirus Disease 2019 (COVID-19). The recently identified coronavirus SARS-CoV-2 poses a threat to worldwide public health. Treatments effective to treat this new virus are urgently needed.

The disclosure is directed to methods of treating COVID-19 in a subject comprising administering to the subject dantrolene or a pharmaceutically acceptable salt thereof or by administering a dantrolene prodrug or a pharmaceutically acceptable salt thereof.

The disclosure is also directed to methods for inhibiting replication of SARS-CoV-2 in a subject comprising administering to the subject dantrolene, or a pharmaceutically acceptable salt thereof or by administering a dantrolene prodrug or a pharmaceutically acceptable salt thereof.

The disclosure is also directed to methods for inhibiting replication of SARS-CoV-2 in a host cell comprising administering to the host cell dantrolene, or a pharmaceutically acceptable salt thereof or by administering a dantrolene prodrug or a pharmaceutically acceptable salt thereof.

The disclosure is also directed to methods for reducing the infectivity of SARS-CoV-2 by administering to a host cell dantrolene, or a pharmaceutically acceptable salt thereof or by administering a dantrolene prodrug or a pharmaceutically acceptable salt thereof.

The present disclosure may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific compositions, devices, methods, applications, conditions, or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure.

As used in the specification including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

When a range of values is expressed, an exemplary embodiment includes from the one particular value and/or to the other particular value. All ranges are inclusive and combinable. Further, reference to values stated in ranges includes each and every value within that range. When values are expressed as approximations, by use of the preposition “about,” it will be understood that the particular value forms another embodiment. The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass reasonable variations of the value, such as, for example, ±10% from the specified value. For example, the phrase “about 50%” can include ±10% of 50, or from 45% to 55%, inclusive of 50%.

It is to be appreciated that certain features of the disclosure which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

As used herein, whether by itself or in conjunction with another term or terms, it should be understood that the phrases “method of treating” and “method of treatment” may be used interchangeably with the phrase “for use in the treatment of” a particular disease.

As used herein, whether by itself or in conjunction with another term or terms, “pharmaceutically acceptable” indicates that the designated entity such as, for example, a pharmaceutically acceptable excipient, is generally chemically and/or physically compatible with other ingredients in a composition, and/or is generally physiologically compatible with the recipient thereof.

As used herein, whether by themselves or in conjunction with another term or terms, “subject(s),” “individual(s),” and “patient(s)”, refer to mammals, including humans. The term human(s) refers to and includes, a human child, adolescent, or adult.

As used herein, whether by themselves or in conjunction with another term or terms, “treats,” “treating,” “treated,” and “treatment,” refer to and include ameliorative, palliative, and/or curative uses and results or any combination thereof. In other embodiments, the methods described herein can be used prophylactically. It should be understood that “prophylaxis” or a prophylactic use or result do not refer to nor require absolute or total prevention (i.e., a 100% preventative or protective use or result). As used herein, prophylaxis or a prophylactic use or result refers to uses and results in which administration of a compound or composition diminishes or reduces the severity of a particular condition, symptom, disorder, or disease described herein; diminishes or reduces the likelihood of experiencing a particular condition, symptom, disorder, or disease described herein; or delays the onset or relapse (reoccurrence) of a particular condition, symptom, disorder, or disease described herein; or any combination of the foregoing.

As used herein, whether used alone or in conjunction with another term or terms, “therapeutic” and “therapeutically effective amount” refer to an amount of a compound or composition that (a) treats a particular condition, symptom, disorder, or disease described herein; (b) attenuates, ameliorates, or eliminates one or more symptoms of a particular condition, disorder, or disease described herein; (c) delays the onset or relapse (reoccurrence) of a particular condition, symptom, disorder, or disease described herein. It should be understood that the terms “therapeutic” and “therapeutically effective” encompass any one of the aforementioned effects (a)-(c), either alone or in combination with any of the others (a)-(c).

As used herein, a “host cell” is, for example, an epithelial cell, for example, a pulmonary epithelial cell, for example, a mammalian pulmonary epithelial cell such as a human pulmonary epithelial cell. Other host cells include white blood cells, for example, macrophages and T-cells.

As used herein, “normalization of fever” is reduction of a subject's temperature to <36.6° C. armpit, <37.2° C. oral, or <37.8° C. rectal, sustained for at least 24 hours.

As used herein, “normalization of oxygen saturation” is an increase in a subject's peripheral capillary oxygen saturation (SpO2) >94%, sustained for at least 24 hours.

As used herein, “inhibiting replication of SARS-CoV-2” refers to decreasing viral load of SARS-CoV-2. Methods for determining SARS-CoV-2 replication inhibition can be determined by those skilled in the art.

Dantrolene is approved for treating malignant hyperthermia and preventing malignant hyperthermia in high-risk patients Malignant hyperthermia is a condition that predisposes susceptible individuals to a life-threatening adverse reaction upon exposure to potent volatile anesthetics (halothane, isoflurane, sevoflurane, desflurane, etc.) and the skeletal muscle relaxant succinylcholine. The anesthetic drugs trigger an uncontrolled Carelease from the endoplasmic reticulum (ER) through the ryanodine receptors (RyR) causing a rapid and sustained rise in myoplasmic Ca. Administration of dantrolene reestablishes cellular calcium homeostasis by inhibiting the release channels in the ER, resulting in lower levels of intracellular Ca.

RYANODEX (dantrolene sodium, 250 mg/vial) is approved for treating malignant hyperthermia and for preventing malignant hyperthermia in high-risk patients. RYANODEX forms an aqueous nanosuspension for IV injection containing dantrolene 50 mg/mL upon reconstitution with 5 mL of USP sterile water for injection (WFI) (without a bacteriostatic agent). Dissolution of RYANODEX suspension in human plasma is extremely rapid, achieving complete dissolution within 1 minute.

Dantrolene is a surprisingly effective treatment for SAR-CoV-2 infection. Dantrolene can decrease the virus' ability to replicate, mature, create virions, release from cells, and/or infect other cells.

Methods of the disclosure can also be accomplished using dantrolene prodrugs, and pharmaceutically acceptable salts thereof. Exemplary dantrolene prodrugs are described in WO2019/079721, the entirety of which is incorporated by reference herein.

Preferred dantrolene prodrugs include, for example, compounds of formula I

wherein R is —P(O)(OH)or —P(O)(OR)(OR); Ris H, —Calkyl, aryl, CalkC(O)O—Calkyl, —CalkOC(O)Calkyl, or CalkOC(O)OCalkyl; and Ris —Calkyl, aryl, CalkC(O)O—Calkyl, —CalkOC(O)Calkyl, or CalkOC(O)OCalkyl, as well as pharmaceutically acceptable salts thereof. Particularly preferred compounds of formula I include compounds 2 and 2a:

Other dantrolene prodrugs include compounds of formula II

wherein Ris H, —C(O)—Z—N(R)(R), —C(O)Z—C(O)—OH, or —C(O)—NH—Y—CH—OC(O)—Z—C(O)—OH; Z is Calk; Y is arylene; Calkyl; Ris H or Calkyl; or Rand R, together with the nitrogen to which they are attached, form a heterocycloalkyl; as well as pharmaceutically acceptable salts thereof.

One aspect of the invention is directed to methods of treating COVID-19 in a subject. The subject may be clinically diagnosed with COVID-19. Criteria for diagnosing a subject with COVID-19 are known and include laboratory confirmation of a SARS-CoV-2 infection as determined using PCR, in combination with presentation of one or more COVID-19 symptoms. Other assays for determining SARS-CoV-2 infections can also be used. Symptoms of COVID-19 include mild to severe respiratory illness with symptoms of fever, cough, and shortness of breath. Some COVID-19 patients may develop pneumonia in one or both lungs. Some COVID-19 patients may develop multi-organ failure.

In some aspects, the subject may be suspected of having COVID-19, based on, for example, having experienced close contact with another person who has been clinically diagnosed with COVID-19 or who has been clinically diagnosed with a SARS-CoV-2 infection. Other subjects may be suspected of having COVID-19 based on the subject's symptom presentation.

In some aspects, the subject is treated for COVID-19 by administering to the subject dantrolene. In other aspects, the subject is treated for COVID-19 by administering to the subject a pharmaceutically acceptable salt of dantrolene, for example, dantrolene sodium. In some aspects, the subject is treated for COVID-19 by administering to the subject a dantrolene prodrug, for example, Compound 2. In some aspects, the subject is treated for COVID-19 by administering to the subject a salt of a dantrolene prodrug, for example, Compound 2a. Administration is preferably of a therapeutically effective amount of the dantrolene, pharmaceutically acceptable salt of dantrolene, dantrolene prodrug, or salt of a dantrolene prodrug. Therapeutically effective amounts include, for example, about 1 mg/kg to 10 mg/kg, administering daily, for one or more days. Particularly preferred amounts include about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 mg/kg, administered daily, in one or more doses, for one or more days.

In some aspects, the administration results in at least a 1-point decrease in the subject's WHO Ordinal Scale score, as compared to the subject's WHO Ordinal Scale score at baseline. Methods of assessing WHO Ordinal Scale score are known in the art. In some aspects, the administration results in a 2-point decrease in the subject's WHO Ordinal Scale score, as compared to the subject's WHO Ordinal Scale score at baseline. In some aspects, the administration results in a 3-point decrease in the subject's WHO Ordinal Scale score, as compared to the subject's WHO Ordinal Scale score at baseline. In some aspects, the administration results in a 4-point decrease in the subject's WHO Ordinal Scale score, as compared to the subject's WHO Ordinal Scale score at baseline. In some aspects, the administration results in a 5-point decrease in the subject's WHO Ordinal Scale score, as compared to the subject's WHO Ordinal Scale score at baseline.

In some aspects, the administration results in an improvement, for example, an increase in the subject's Sequential Organ Failure Assessment daily score, as compared to baseline. Methods of assessing a subject's Sequential Organ Failure Assessment daily score are known in the art.

In some aspects, the administration results in a reduction of time to normalization of fever in the subject, as compared to the amount of time to normalization of fever in a control subject, for example, as compared to a subject who has only received standard of care treatment. In some aspects, the administration results in a reduction of fever in the subject treated for COVID-19. In other aspects, the administration results in a clinically significant reduction of fever in the subject. In some aspects, the administration results in a normalization of fever in the subject.

In some aspects, the administration results in a reduction of time to normalization of oxygen saturation in the subject, as compared to the amount of time to normalization of oxygen saturation in a control subject, for example, as compared to a subject who has only received standard of care treatment. In some aspects, the administration results in an increase of oxygen saturation in the subject. In other aspects, the administration results in a clinically significant increase in oxygen saturation in the subject. In other aspects, the administration results in normalization of oxygen saturation in the subject.

In yet other aspects, the administration results in improvement in one or more symptoms of COVID-19 in the subject. In other aspects, the administration results in a clinically significant improvement in one or more symptoms of COVID-19 in the subject.

Other aspects of the disclosure are directed to methods of inhibiting replication of SARS-CoV-2 in a subject. The subject may be clinically diagnosed with a SARS-CoV-2 infection. Criteria for diagnosing a subject with a SARS-CoV-2 infection are known and include laboratory confirmation as determined using PCR. Other assays for determining SARS-CoV-2 infections can also be used.

In some aspects, the subject may be suspected of having a SARS-CoV-2 infection, based on, for example, having experienced close contact with another person who has been clinically diagnosed with COVID-19 or who has been clinically diagnosed with a SARS-CoV-2 infection.

In some aspects, inhibition of SARS-CoV-2 replication in a subject is accomplished by administering to the subject dantrolene. In other aspects, inhibition of SARS-CoV-2 replication in a subject is accomplished by administering to the subject a pharmaceutically acceptable salt of dantrolene, for example, dantrolene sodium. In some aspects, inhibition of SARS-CoV-2 replication in a subject is accomplished by administering to the subject a dantrolene prodrug, for example, Compound 2. In some aspects, inhibition of SARS-CoV-2 replication in a subject is accomplished by administering to the subject a salt of a dantrolene prodrug, for example, Compound 2a. Administration is preferably of a therapeutically effective amount of the dantrolene, pharmaceutically acceptable salt of dantrolene, dantrolene prodrug, or salt of a dantrolene prodrug. Therapeutically effective amounts include, for example, about 1 mg/kg to 10 mg/kg, administering daily, for one or more days. Particularly preferred amounts include about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 mg/kg, administered daily, in one or more doses, for one or more days.

Some aspects of the disclosure are directed to methods for inhibiting replication of SARS-CoV-2 in a host cell. Inhibition of viral replication can be determined by those skilled in the art. In these methods, replication is inhibited by administering dantrolene to the host cell. In other aspects, replication is inhibited by administering a pharmaceutically acceptable salt of dantrolene to the host cell, for example, dantrolene sodium. In some aspects, replication is inhibited by administering to the host cell a dantrolene prodrug, for example, Compound 2. In some aspects, replication is inhibited by administering to the host cell a salt of a dantrolene prodrug, for example, Compound 2a.

Some aspects of the disclosure are directed to methods for inhibiting entry of SARS-CoV-2 into a host cell. Inhibition of viral entry into a host cell can be determined by those skilled in the art. In these methods, viral entry is inhibited by administering dantrolene to the host cell. In other aspects, viral entry is inhibited by administering a pharmaceutically acceptable salt of dantrolene to the host cell, for example, dantrolene sodium. In some aspects, viral entry is inhibited by administering to the host cell a dantrolene prodrug, for example, Compound 2. In some aspects, viral entry is inhibited by administering to the host cell a salt of a dantrolene prodrug, for example, Compound 2a.

Some aspects of the disclosure are directed to methods for inhibiting SARS-CoV-2 virion maturation in a host cell. Inhibition of virion maturation in a host cell can be determined by those skilled in the art. In these methods, virion maturation is inhibited by administering dantrolene to the host cell. In other aspects, virion maturation is inhibited by administering a pharmaceutically acceptable salt of dantrolene to the host cell, for example, dantrolene sodium. In some aspects, virion maturation is inhibited by administering to the host cell a dantrolene prodrug, for example, Compound 2. In some aspects, virion maturation is inhibited by administering to the host cell a salt of a dantrolene prodrug, for example, Compound 2a.

Some aspects of the disclosure are directed to methods for release of SARS-CoV-2 from a host cell. Inhibition of release from a host cell can be determined by those skilled in the art. In these methods, viral release is inhibited by administering dantrolene to the host cell. In other aspects, viral release is inhibited by administering a pharmaceutically acceptable salt of dantrolene to the host cell, for example, dantrolene sodium. In some aspects, viral release is inhibited by administering to the host cell a dantrolene prodrug, for example, Compound 2. In some aspects, viral release is inhibited by administering to the host cell a salt of a dantrolene prodrug, for example, Compound 2a.

Some methods of the disclosure are directed to methods for reducing the infectivity of SARS-CoV-2. Reduction of infectivity can be determined by those skilled in the art. In these methods, infectivity is reduced by administering dantrolene to the host cell. In other aspects, infectivity is reduced by administering a pharmaceutically acceptable salt of dantrolene to the host cell, for example, dantrolene sodium. In some aspects, infectivity is reduced by administering to the host cell a dantrolene prodrug, for example, Compound 2. In some aspects, infectivity is reduced by administering to the host cell a salt of a dantrolene prodrug, for example, Compound 2a.

The following examples are provided to illustrate some of the concepts described within this disclosure. While each example is considered to provide specific individual embodiments of disclosure, none of the Examples should be considered to limit the more general embodiments described herein. In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental error and deviation should be accounted for.

Methodology

The study is a single-center, open-label, two-arm parallel study of dantrolene for the adjuvant treatment of COVID-19 administered intravenously (IV). In one treatment arm, dantrolene will be administered in conjunction with current standard of care following medical practice and procedures established for the in-hospital treatment of patients with COVID-19. In the second treatment arm, subjects will receive current standard of care following medical practice and procedures established for the in-hospital treatment of patients with COVID-19.

Following initial triage and primary assessment of a subject, the subject's baseline status will be documented. Once eligibility criteria and baseline status are obtained, administration of dantrolene will be initiated.

Eligible COVID-19 subjects randomized to Group A will receive dantrolene (as RYANODEX, dantrolene sodium) as follows: